Anticoccidial method

ABSTRACT

1. A METHOD OF TREATING AND PREVENTING COCOCIDIOSIS IN POULTRY AND RABBITS WHICH COMPRISES ADMINISTERING ORALLY TO POULTRY AND RABBITS AN ANTICOCCIDALLY-EFFECTIVE AMOUNT OF ANTIBIOTIC SELECTED FROM THE GROUP CONSISTING OF A28695, A28695A, A28695B, AND THE SODIUM SALT, POTASSIUM SALT, SODIUM-POTASSIUM MIXED SALT, OR AMMONIUM SALT THEREOF.

Oct. 1, 1974 Filed July 1a. 1973 INFARED ABSORPTION SPECTRUM OFANTIBIOTIC A28695 A SALT FREQUENCY (cw) I5OOL4OOI3OO I200 uoo R. I...HAMILL EFAL ANTICOGCIDIAL METHOD 9 1'0 WAVELENGTH (M ICRONS)NOISSIWSNVELL .LNBDHHd Fig.

2 Sheets-Sheet 1 United States Patent Office 3,839,559 ANTICOCCIDIALMETHOD Robert L. Hamill, New Ross, and Marvin M. Hoehn, Indianapolis,Ind., assignors to Eli Lilly and Company, Indianapolis, Ind.Continuation-impart of application Ser. No. 211,231, Dec. 23, 1971, Thisapplication July 16, 1973, Ser.

Int. Cl. A61k 21/00 U.S. Cl. 424-121 6 Claims ABSTRACT OF THE DISCLOSUREMethods and compositions for the control of coccidiosis usingantibiotics A28695A and A28695B, or the mixture thereof, identified asA28695, and their physiologically-acceptable salts as the activeanticoccidial agent.

CROSS-REFERENCE This application is a continuation-in-part of copendingapplication Ser. No. 211,231, filed Dec. 23, 1971.

BACKGROUND OF THE INVENTION 1. Field of the Invention attacks the smallintestine of the chick causing intestinal coccidiosis. E. meleagrimitisand E. adenoeides are causative organisms of coccidiosis in turkeys.

In rabbits (Oryctolagus cuniculus), coccidiosis is caused by one or moreof the following species of the genus Eimeria. Thus, E. stiedae attacksthe liver, while E. ceci cola, E. intestinalis, E. irresidua, E. media,E. magna, E. perforans, E. piriformis, and E. neoleporis, all occur insome portion of the intestinal tract of the rabbit.

When left untreated,'the severe infections of coccidiosis lead to poorweight gain, reduced feed efficiency and high mortality in fowl orrabbits. The morbidity and mortality occasioned by coccidiosisinfections create extensive economic loss when such infections are leftuntreated or unchecked. The elimination or control of this disease is,

therefore, of paramount importance to the poultry and rabbit raisingindustry.

2. Description of the Prior Art In the prior art, Gorman et al., U.S.3,55,150 (Ian. 12, 1971) teach the use of the antibiotic nigericin, itsalkali metal, alkaline earth, and basic nitrogen salts for theprevention and treatment of coccidiosis in poultry, including chickensand turkeys.

Further, Gorman et al., U.S. 3,577,531 (May 4, 1971) teach the use ofthe antibiotic dianemycin and its alkali metal, alkaline earth, andbasic nitrogen salts'in the prevention and treatment of coccidiosis inchickens and turkeys.

In addition, Gorman et al., U.S. Pat. 3,627,883 (Dec. 14, 1971) teachthe use of antibiotic X-206, its alkali metal, alkaline earth metal, orbasic nitrogen salts as potent poultry coccidiostats. I

Bloss, U.S. Pat. 3,482,023 (Dec. 2, 1969), teaches the cidiosis inmeat-producing animals.

3,839,559 Patented: Oct. 1, 19 74 Also in the prior art, Fitzgerald, J.Protozool 19 (2) 332-334 (1972) discusses the etiicacy of the antibioticmonensin in the treatment of hepatic coccidiosis in rabbits. Further inthe prior art is Haney et al., U.S. Pat. 3,501,- 568 (Mar. 17, 1970),With claims to the antibiotic A3823, also known as monensin, and whichpatent teaches the use of antibiotic A3 823 complex to prevent thedevelopment of coccidiosis in poultry.

Berger, Ger. Otfen. 2,140,322 (Feb. 17, 1972), teaches the antibioticX-5l08 and its activity against gram-negative and gram-positivebacteria, as well as activity against streptococci, Penumococci, andcaecal coccidiosis.

Also in the prior art, Kubota et al., Japanese 72 01,288 (Jan. 13,1972), teach the preparation of polyetherin A 30-acylates havingactivity against Eimeria tenella and Eimeria acervulina. And further,Kubota et al., Japanese 72 14,224 (Apr. 27, 1972), teach the preparationof polyetherin A 29-ethers which are useful for the control ofcoccidiosis in chickens caused by Eimeria tenella or Eimeria acervulina.

And further in the prior art, Hamill e1 al., U.S. 3,705,- 238 (Dec. 5,1972), teach antibiotics A2041 and A204II and methods for theirproduction, the antibiotics being taught as having anticoccidial,insecticidal, anti-PPLO, and antimicrobial activity.

Berger, U.S. 3,719,753 (Mar. 6, 1973), teaches the use of antibioticX-537A in compositions for the treatment and prevention of coccidiosisin poultry.

Although a number of antibiotics have been used in compositions andmethods for the control of coccidiosis, there remains a need for moreeffective and economicallyuseful compositions and methods for thistreatment.

SUMMARY A method for the control and/or prevention of coccidiosis,together with novel compositions useful in that method, which method andcompositions empoly antibiotics A28695, A28695A, A28695B, and saltsthereof are disclosed.

I DETAILED DESCRIPTION This invention relates to the use of certainacidic, nonnitrogenous antibiotics and the alkali metal, alkaline earthmetal, and ammonium salts thereof as anticoccidial agents. Moreparticularly, the invention relates to the use of antibiotics A28695Aand A28695B, or the mixture thereof, identified as A28695, in a novelmethod and in novel compositions for the control and/ or prevention ofcoccidiosis in poultry and in rabbits.

In the treatment of coccidiosis infections, relatively low levels of theA28695 antibiotics in poultry feed are sufficient to afford the poultrygood protection against coccidiosis. Thus, one of the antibiotics, orthe mixture, is administered to chickens in an amount equal to about0.00125 percent to 0.03 percent by weight of the daily feed intake. Thepreferred range for antibiotic A28695A is from about 0.0025 to about0.01 percent, with the optimum results being obtained when from about0.0025 to about 0.005 percent (22.7-45.4 g./ton) of antibiotic A28695A,or 00.02 percent (181.6 g./ton) of antibiotic A28695B'is incorporatedinto the poultry feed. The mixture, antibiotic A28695, is effective atfrom about 0.0025 percent to about 0.02 percent of the daily feedintake.

In addition tothe utility of antibiotics A28695 as anticoccidial agentsin poultry, it has also been determined that antibiotic A28695A isactive as an anticoccidial agent in rabbits when administered in anamount equal to about 0.00125 percent to about 0.005 percent by Weightof the daily' feed intake, while about twice that amount of antibioticA28695B is required to accomplish the same results.

designated A28695A and A28695B. They are produced,

8 more accurate, becauseof al'ong'with other unidentified antibiotic"substances, by

culturing the microorganism Streptomyces albus NRRL 3883 in an aqueous,nutrient culture medium under submerged aerobic fermentation conditionsuntil a substantial level of antibiotic activity is produced. Antibioticfactor A28695A is produced in greater abundance than is antibioticA28695B. The other antibiotic substances produced in the fermentationoccur in such minor amounts as to render their recovery unrewarding. 4

Antibiotic A28695A, as'isolated from the A28695 mixture of antibiotics,is obtained as a White, crystalline mixed sodium-potassium salt having amelting point of about 161l65 C.

' The mixed sodium-potassium salt of antibiotic A28695 A is insoluble inwater, slightly soluble in methanol, soluble in ether, and soluble inesters such as methyl acetate, ethyl acetate, and the like; ketones suchas acetone and methyl ethyl ketone; the halogenated hydrocarbons such Has chloroform; and the aromatic hydrocarbons such as benzene andtoluene. Antibiotic A28695A, as the sodiumpotassium salt, is stable insolution at pH values above pH 4.0 at temperatures up to about 27 C. Thespecific optical rotation, [a] of the mixed sodium-potassium salt ofantibiotic A28695A is +14.07 (C=1, methanol).

The' infrared absorption spectrum of antibiotic A28695A, as the mixedsodium-potassium salt, in chloroform solution, is shown in FIG. 1 of theaccompanying drawings. The following distinguishable absorption maximain the spectrum are observable over the range of 2.0 to 15.0 microns:3.1-3.3, 3.4, 3.47, 6.24, 6.84, 7.00, 7.25, 7.37, 7.49, 7.68, 7.78, 8.1,8.47, 8.61, 8.95, 9.11, 9.20, 9.42, 9.5, 9.80, 9.98, 10.24, 10.54,10.87, 11.09, 11.5, and 11.66 microns. The antibiotic A28695A has nocharacteristic ultraviolet absorption pattern.

A powder X-ray diffraction pattern of the crystalline mixedsodium-potassium salt of antibiotic A28695A, using vanadium-filteredchromium radiation, and a wave length value of 2.2896 A for calculatingthe interplanar spacings, gives the following values:

The free acid of A28695A is a white crystalline solid melting at about9799 C. Elemental analysis of the free acid form of antibiotic A8695Agives the following elemental composition: 63.31 percent carbon; 8.83per-' cent hydrogen, and 28.03 percent oxygen. Mass spectralthan thevalue from the mass spectral data.'The value' calculated from the massspectral data is probably the the limitations of the titration method.Nuclear magnetic resonance spectral data indicate the presence of fourmethoxy groups in antibiotic A28695A.

The mixed sodium-potassium salt of antibiotic A28695B is a white,crystalline compound melting at about 172 C. The solubility andstability pattern of the antibiqticfis similar to that of the mixedsodium-potassium salt of antibiotic A28695A. The specific opticalrotation, [a] of the mixed sodium-potassium salt of antibiotic A28695Bis +10.1' (C=l, methanol).

The infrared absorption spectrum of A28695B, as the mixedsodium-potassium salt in chloroform solution, is shown in FIG. 2 of theaccompanying drawings. The distinguishable bands in the infraredspectrum over the range of 2.0 to 15.0 microns are as follows: 3.0, 3.4,3.47, 6.24, 6.85, 701,726, 7.3, 7.68, 7.78,'8.1, 8.58, 8.82, 8.95, 9,11,9.19, 9.45, 9.59, 9.82, 10.04, 10.28, 10.55, 11.10, 11.24 and 11.65microns. The antibiotic A28695B has no characteristic ultravioletabsorption pattern.

The acid form of antibiotic A28695B is a white crystalline solid with amelting point of about 122-124 C. Microanalysis gives the followingpercent elemental composition of the acid form of A28695B: 60.49 percentcarbon; 9.15 percent hydrogen, and 31.32 percent oxygen.

Nuclear magnetic resonance spectral data indicate that antibioticA28695B contains three methoxy groups. Mass spectral data on antibioticA28695B indicate an approximate molecular weight of 846. Electrometrictitration of antibiotic A28695B, as the sodium salt in 66 percentaqueous ethanol, shows the presence of one titratable group having a pKavalue of 5.9. The molecular weight of the sodium salt of antibioticA28695B, calculated from the titration data, is approximately 877. Themolecular weight of the free acid of antibiotic A28695B would thereforebe approximately 855.

A powder X-ray diffraction pattern of the crystalline antibiotic A28695Bacid, using vanadium-filtered chromium radiation, and a wave lengthvalue of 2.2895 A for calculating interplanar spacings, gives thefollowing values:

d I/I d I/I, 13.54 .50 3.98 .10 12.63 .05 3.84 .60 11.52 .15 3.73 .059.96 .02 3.66 .05 9 .39 .60 3.57 .05 7.88 .20 3.48 05 7.52 .20 3.22 157.08 .30 3.07 10 6.66 1.00 3.05 .02 6.46 .20 2.94 .02 6.28 .20 2.84 .026.05 .30 2.72 .10 5.81 .50 2.56 02 5.57 .20 2.33 .02 5.33 .70 2.27 024.92 .60 2.15 .05 4.63 .60 2.09 .02 4.51 .20 2.07 .02 4.29 .30 2.03 .024.14 .30

The paper chromatographic behavior of the mixed sodium and potassiumsalts of A28695A and B is shown by the Rf values in Table I below. Thevalues were obtained in the indicated solvent systems, using in eachinstance Whatman No. 1 paper. The location of the antibiotics on thechromatogram was determined by bioautograph using Bacillus subtilis asthe detecting organism.

to color tabs in the Tresner and Backus color series TABLE I [Appl.Microbiol. 11:335-338 (1963)]. The Maerz and hr t h of antibioticsA28695A and A28695B Papem oma ogmp y Paul color block designations,[Dictionary of Color, Mc- Rfvalu" Graw-Hill Book Co., Inc., New York(1950)], are en- Solvent system A28695A 'A28695B 5 closed in brackets.'ISP numbers refer to International Stre t Water saturated with butanol0 53 33 p omyce prolect fi shlrhngand gq (avall Water saturated withbutanol; 2% tolu 64 9 able from Difco Laboratories, Detroit, Michigan).Obsersulfonic acid; 17 piperidine V a water saturated vgith methylisobutyl ketone; vations were made follow ng incubation at 30 C. for

2% p-toluene sulfonic acid; 1% piperidine 0. 58 0. 74 fourteen daysunless otherwise noted. Waterxrnethanolzacetone (12:3:1)*".. 0.25 0.54Benzene saturated with water 0.57 0.48 icr sc pic Morphology, CulturalCharacteristics, and Physiology i val e is defined as the ratio of thedistance traveled by the an i- Microscopic morphology Sporophores arespiralled. Spores biotic from the origin to the distance traveled by thesolvent front from are oval (LO-1.25;; x 0.5-1.0 the origin. i and occurin chains of 10 to 50.

"This solution is adjusted to pH 10.5 with NHrOH and then the pH 15Cultural characteristics: lowered to pH 7.5 with H3PO4- ISP ed u #2Abundant growth; reverse light,

' i h 15 yellofv brov\1r1n[l1E5(]1. Good1 9.8 a myce 111118.11 SDOIII 8-Thrn-layer chromatography on silica gel plateswit a tion'white (WM'NOSoluble vanillin spray as a detecting agent is also vused to identify I#3 i r wt h i l SP medium Fa gro ;reverse white. Fa r and separateant1biot1c A2869 5A and B. The chromato aerialmycelmm and sperm? graphicbehavior on silica gel 18 shown below. on white (wmywith Scattered 20areas of light yellow, (Y) 1% fl:v TABLE II ISP medium #4 Abu1r1idan[t1(gi oswtni) levglset pall iaa1 ye OW 11H an ac Thin-layer chromatographyof antibiotics A28695A and A28695B mycehum and spomlation,

white (W)a. Light brown Rf Value I dl o ii i i ti h SP me um 5 00 0a unant growt ;reverse Solve system A28695A M86953 light yellow [10m Good toabundant aerial mycelium and Benzenezethyl acetate O- 71 61 spores, paleyellow (Y):Eba. No Chloroformzethyl acetate (2:3) 0. 69 0. 61 solublepigment Benzeneiacetone 29 20 Calcium malate Good growth; reverse lightyellow [10F3]. Moderate aerial mycelium and sporulation, pale yellow TheA28695 antibiotics have the ability to form comange s. Nonetgshghtyellow S0 11 e 1 men plexes with monovalent CatlOIlS- In eXPenmentS t030 c k medium Good gr0wPt%;reVerse light yellow mine ion specificity,antibiotic A28659A showed specific- 9131. G23? er i al lgllycelilllim3.1150 8.10, 680W ity for potassium ions and rubidium ions, whileantibiotic (YWEL N0sO1ubepigYment A28695B showed specificity for sodiumand potassium Tomato paste oatmeal Abufidanhg ggtharevderse ipazileions. The use of ion-specific electrodes is important 1n s r gfi i- 1 2a a many chemical analyses. Because of the unique properties,ye1l111%v1vishigIrIitE%G)2dc. No antibiotics A28695A and B are suitableas components of Physiology: 50 P g Temperature requirements"... Goodgro i th and sporulation at Ion Speclfic. eieqtrodes d d ac -37 o. Nogrowth at 43 0.,

The antibiotics useful in this invention are pro uce 4 0 CI,

tr in Of an actinom cete Skim milk; No curd or clearing after 21 days.by cul-turmg newly dlscovered a y t 40 Surface ring of growth; sediment.organism under subm rg aere 10 Con gh In a Gelatin completenquefamonafter 21 ent culture medium until the culture me win contains days.

b t ti l antibiotic activity. The antibiotics can be re- Nltmte reductwnSlight reductim after 21 days" covered from the fermentation medium byemploying various isolation and purification procedures known in theart. Table VI Summarizes the lts f the Carbon utiliza- The actinomyceteused in the production ofrthe' anti tion tests carried out on theA28695-produc1ng culture,

biotics useful in this invention has been identified as a strain ofStreptomyces albus (Rossi-Doria) Waksman and Henrici. The organism hasbeen deposited without restriction as to availability with the permanentculture collection of the Northern Utilization Research and DevelopmentDivision, Agricultural Research Service, United States Department ofAgriculture, Peoria,.Illinois. Its accession number in this collectionis NRRL 3883. The strain was isolated from a soil sample collected inCuracao (Dutch Antilles). Portions of the soil sample were suspended insterile deionized water and the suspensions were streaked on nutrientagar in Petri plates. After incubation at 25 -35 C. until growth wasattained, colonies of the antibiotic A28695-producing organisms weretransferred to agar slants with a sterile platinum loop. The agar slantswere then incubated to provide a suitable inoculum for the production ofA28695.

The methods employed in the taxonomic studies of the A28695-producingculture, NRRL 3883, were those recommended for the InternationalStreptomyces Project [Shirling and Gottlieb, Intern. Bull. SystematicBacterioL, 16:313-340 (1966)], together with certain supplementarytests. Results of the taxonomic studies are summarized in the paragraphswhich follow. Color names were assigned according to the Inter-SocietyColor Council-National Bueau of Standads (ISCC-NBS) method (KellyandJudd, The ISCC-NBS Method of Designating Colors a nd a Dictionary ofColor Names, U.S. Dept. of Commerce Circ. 553, Washington, DC, 1955).Letters in parentheses refer to color blocks and underlined letters andnumbers NRRL 3883. Symbols employed in the table are as follows:

+qositive utilization probable utilization )=questionable utilization-=no utilization The A28695-producing culture (NRRL 3883) appears to bevery similar to the strain of Streptomyces aIbus ATCC 3004 as describedby Lyons and Pridham, J. BacterioL, 83:370-380 (1962). Variations occurin the utilization of four carbon sources and in growth above 37 C. TheNRRL 3883 culture employed in this invention is also similar to NRRL3384, which produces antibiotic A204 [Belgian Pat. No. 728,382(8-1369)]; observed differences indicate that NRRL 3384 produces slightlylonger spores, does not liquefy gelatin, and grows at somewhat highertemperatures.

The culture medium employable in producing antibiotics A28695A and B bycultivation of the above-described organism can be any one of severalmedia, since, as is apparent from the above-described utilization tests,the organism is capable of utilizing different energy sources. However,for economy of production, maximum yield of antibiotic, and ease ofisolation of the antibiotic, certain culture media containing relativelysimple nutrient sources are preferable. For example, the media useful inthe production of antibiotics A28695A and B include an assimilablesource of carbon such as glucose, mannitol, fructose, soluble starch,dextrin, molasses, brown sugar and the like. Preferred sources of carbonare glucose and dextrin. Additionally, employable media include a sourceof assimilable nitrogen such as oatmeal, beef extract, hydrolyzedcasein, corn steep liquor, yeast extract, soybean meal, peptones (meator soy) and the like. Preferred sources of nitrogen are soybean meal andacid-hydrolyzed casein.

Mineral salts, for example those providing calcium, magnesium, sodium,potassium, cobalt, chloride, sulfate and carbonate ions, and a source ofgrowth factors, such as yeast or yeast extract, can be incorporated intothe media with beneficial results.

As with many microorganisms, it is believed to be desirable to includethe so-called trace elements in the culture medium for growing theactinomycete employed to produce the antibiotics used in this invention.Such trace elements are commonly supplied as impurities incidental tothe addition of the other constituents of the medium.

Production of the antibiotics used in this invention can be effected atany temperature conducive to satisfactory growth of the microorganism,for example, between about 26 C. and 40 C. and preferably between about2630 C. Ordinarily, optimum production of the antibiotics is obtained inabout two to five days.

The initial pH of the culture medium can be varied widely. However, ithas been found desirable that the initial pH of the medium be betweenabout pH 6.5 and about pH 7.2. As has been observed with otheractinomycetes, the pH of the medium gradually increases throughout thegrowth period of the organism and may attain a level of from about pH7.0 to about pH 8.0 or above. The final pH is dependent at least in parton the initial pH of the medium, the buffers present in the medium, andthe period of time the organism is permitted to grow. Small quantitiesof the antibiotic are obtained conveniently by the use of shake flasksand by surface culture in bottles. For the production of substantialamounts of antibiotic A28695, however, submerged aerobic culture inlarge tanks is preferably employed.

In order to avoid a pronounced lag in the production of the antibioticwith the attendant inefficient utilization of equipment, it is preferredto use the vegetative rather than the spore form of the organism forinoculation of the medium in the production tanks. Accordingly, avegetative inoculum or the organism is first prepared by inoculating arelatively small quantity of the culture medium with the spore form ofthe organism, and the young, active vegetative inoculum so obtained isthen transferred aseptically to the large production tanks. The mediumin which the vegetative inoculum is produced can be the same as thatutilized for the production of the antibiotic, although other media canbe employed advantageously.

As is customary in submerged aerobic culture processes, sterile air isblown through the culture medium. For efficient growth of the organismand antibiotic production, the volume of air employed in the tankproduction of antibiotics A28695A and A28695B is preferably about 1volume of air per minute per volume of culture medium. Efficient growthand optimal yields of antibiotics A28695A and A28695B are obtained whenthe volume of air used is at least three-tenths volume of air per minuteper volume of culture medium.

The concentration of antibiotic activity in the culture medium can befollowed readily during the fermentation period by testing samples ofthe culture medium for their inhibitory activity against the growth ofan organism known to be inhibited in the presence of antibiotics A28695Aand A28695B. The use of the organism Bacillus subtilis has been found tobe suitable for this purpose. The testing can be carried out bywell-known turbidimetric or disc plate assay methods.

A variety of procedures can be used in the isolation and purification ofantibiotics A28695A and A28695B, for example, solvent extraction, anduse of adsorbents and chromatography columns. Solvent extractionprocedures are preferred for commercial production inasmuch as they areless time consuming and less expensive, and higher recovery yields areobtained thereby.

The antibiotic activity is located in the mycelium as well as in thefermentation beer. The mycelium can be separted from the fermentationbeer by filtration with the use of a filter aid, and both the mycelialcake and the filtered fermentation medium extracted with a suitable organic solvent to recover the A28695 activity. Alternatively, theunfiltered fermentation beer can be extracted with an organic solvent torecover the antibiotic activity. Suitable extraction solvents include,for example, ethyl acetate, amyl acetate, ethanol or methanol. Theantibioticextracts are evaporated under reduced pressure to obtain animpure mixture of the A28695 antibiotics as an oily residue. Theantibiotics thus recovered are present in the form of their mixedsodium-potassium salts. Further purification of the antibiotic mixturecan be accomplished by chromatography of the oily residue over asuitable adsorbent such as activated carbon or silica gel. An activatedcarbon adsorbent such as Pittsburgh Carbon is a preferred adsorbent forpurifying the A28695 antibiotic mixture.

The individual antibiotics can be separated from the mixture by furtherchromatography. Thus, for example, the mixture of the sodium-potassiumsalts of A28695A and B can be dissolved in a solvent system consistingof benzenezethyl acetate (9:1) and the solution so obtainedchromatographed on a column packed with silica gel. The column is theneluted with the same solvent mixture and multiple fractions arecollected. The progress of the fractionation is monitored by examiningthe individual fractions on thin-layer chromatograms or paperchromatograms. The fractions containing each individual antibiotic arecombined and the solvent is removed by evaporation to yield, insubstantially pure form, the separated antibiotics in the form of theirmixed sodium-potassium salts.

The preparation of antibiotics A28695A and A28695B is furtherillustrated by the following examples.

EXAMPLE 1 A. Shake-flask fermentation of A28695 The A28695-producingculture is prepared and maintained on an agar slant having the followingcomposition:

Dextrin 700 g 10.0 NZ amine A 2 g 2.0 Beef extract g 1.0 Yeast extract g1.0 Agar g 20.0

Deionized water 1 Potato dextrln imported from Holland. Shel-heldChemical 00., Division of National Dairy Products Corp., Norwich, N.Y.

The slant is inoculated with the A28695-producing culture, NRRL 3883,and incubated at 30 C. for about 4-6 days. The sporulated slant iscovered with a small amount of sterile deionized water and gentlyscraped to provide an aqueous spore suspension.

One milliliter of the resulting spore suspension is used to inoculate100 ml. of sterile vegetative medium having the following composition:

The inoculated vegeative medium is incubated for about 24-48 hours at 30C. on a reciprocal shaker having a twoinch stroke operating at 108strokes per minute, or on a rotary shaker operating at 25 r.p.m. A m1.portion of the resulting culture is then employed to inoculate 100 ml.of sterilized production medium contained in a 500 ml. Erlenmeyer flaskand having the following composition:

Soybean meal g 15.0 Casein g 1.0 NaNO3 g. 3.0 Glucose syrup g 20.0 Tapwater l 1 The inoculated production medium is allowed to ferment for42-72 hours at 2530 C. on either a rotary shaker operating at 250r.p.m., or on a reciprocal shaker operating at 108 strokes per minute.The observed terminal pH is between about pH 6.5 and pH 8.0.

B. Tank fermentation of A28695 The A28695-producing culture is preparedand main tained on an agar slant having the following composition:

Dextrin g 10.0 Yeast extract g 1.0 Enzyme-hydrolyzed casein g 2.0 Beefextract g 1.0 COC12'6H20 g Agar g 20.0 Deionized water l 1 Glucose g15.0 Soybean grits g 15.0 Corn steep liquor g 10.0 NaCl g 5.0 C-aCO g2.0 Tap water liters 1.1

The pH of the medium is adjusted to pH 6.5 with sodium hydroxidesolution and is unchanged by sterilization by autoclaving at 15-20pounds pressure for 30 minutes.

The inoculated, sterilized medium is allowed to ferment for about 72hours at 30 C. on a rotary shaker operating at 250 r.p.m. A 10 ml.portion of the resulting culture is used to inoculate 200 ml. ofsterilized secondstage growth medium contained in a liter flask andhaving the same composition described above.

The inoculated medium is allowed to ferment for about 30 hours at about30 C. on a reciprocal shaker operatingat 250 r.p.m. A 200 ml. portion ofthe resulting culture is used to inoculate liters of the followingmedium in a 40-li'ter fermentor.

Ingredient: Percent Glucose 2.5 Soybean grits 1.5 Acid-hydrolyzed casein0.1 Molasses 0.3 CaCO 0.25 Tap water 25 liters The pH of the medium isabout pH 7.2 after sterilization by autoclaving at l520 pounds pressurefor 30 minutes.

The inoculated medium is aerated at a rate of one volume of air pervolume of culture per minute and is stirred with conventional agitatorsat 350 r.p.m.

The fermentation is carried out at about 30 C. for about 5 days.

C. Isolation of Antibiotic Mixture Ninety-two liters of wholefermentation broth obtained from an A28695 fermentation was ifilteredwith the aid of a commercial filter aid. The mycelial cake was suspendedin 25 liters of methanol, and the mixture was stirred vigorously forabout 30-60 minutes. The mixture was then filtered, and the filtrate wasconcentrated to remove the methanol. The aqueous phase so obtained wascombined With the filtrate from the original fermentation broth.

The extracted mycelial cake was then suspended in 25 liters of ethylacetate and the suspension was stirred for about 30-60 minutes. Themixture was then filtered, and the mycelial cake was discarded. Theethyl acetate extract of the mycelial cake was saved to combine with theethyl acetate extracts of the filtered broth. The filtered broth wasthen extracted twice with half volumes of ethyl acetate. The spent brothwas discarded. The ethyl acetate extracts of the broth were combinedwith the ethyl acetate extract of the mycelial cake.

Alternatively, the A28695 activity was extracted from the unifilteredfermentation medium by the following procedure: Ninety-two liters of thewhole fermentation medium was stirred with an equal volume of ethylacetate. The mixture was filtered and the resulting filtrate wasseparated into the ethyl acetate phase and the aqueous phase. Theaqueous phase was discarded, and the ethyl acetate phase was saved to becombined with a second extract.

The extracted mycelial mass was then extracted a second time with ethylacetate. The mycelial mass was discarded. The ethyl acetate extract wascombined with the original extract.

The pooled extracts, obtained by extraction of either whole fermentationmedium, or of the broth and mycelium separately, were concentrated to anoily residue. This oily residue was dissolved in one liter ofchloroform. The chloroform solution was passed over a 5.5 cm. x cm.column of Pittsburgh Carbon (12 x 40 mesh) packed in chloroform. Thecolumn was washed with 20 liters of chloroform. The chloroform efiiuentand wash were combined and concentrated to a dry residue. Seventy andfour-tenths grams of A28695 activity was recovered.

D. Separation of Antibiotics A28695A and A28695B Thirty grams of crudeantibiotic mixture A28695, obtained according to the procedure describedin the previous section, was dissolved in a 9:1 mixture of benzene andethyl acetate. The solution was passed over a 5.5 cm. X cm. column ofsilica gel (Grace grade number 62, Davison Chemical, Baltimore, Md.21226). The adsorbent had previously been washed with benzenesethylacetate (9:1). The column was washed with six liters of benzenezethylacetate (9:1) and the effluent and wash were discarded. The column wasthen eluted with a benzene:ethyl acetate solution (4:1). The eluate wascollected in multiple fractions, antibiotic A28695A coming off thecolumn in the early fractions, while antibiotic A28695B was collected insubsequent fractions. The

identity of the antibiotic in the respective column fractions wasdetermined by paper chromatography and thinlayer chromatography. Thecolumn fractions containing the same antibiotic were combined andevaporated in vacuo to yield the respective individual antibiotics insubstantially pure form.

Antibiotic A28695A was crystallized by dissolving the amorphousantibiotic in warm ether. The antibiotic crystallized as the mixedsodium-potassium salt with a melting point of about 163-165 C. Yield:11.8 grams.

Antibiotic A28695B was also crystallized from ether in the form of themixed sodium-potassium salt with a melting point of about 152-154" C.Yield: 5.3 grams.

EXAMPLE 2 Preparation of Acid Form of Antibiotic A28695A Five grams ofthe mixed sodium and potassium salt of antibiotic A28695A was dissolvedin 105 ml. of dioxane. Forty ml. of water was added to the solution. ThepH of the solution was adjusted to pH 4 with hydrochloric acid. Thesolution was concentrated in vacuo to remove the dioxane. The resultingaqueous solution was extracted twice with an equal volume of ethylacetate and the spent aqueous phase was discarded. The ethyl acetateextracts were combined and were concentrated to dryness. The driedresidue was dissolved in warm ethyl ether. The ether solution waschilled overnight to allow antibiotic A28695A to crystallize. Thecrystals were recovered by filtration and dried. Yield: 4.5 g. M.P.97-99 C.

EXAMPLE 3 Preparation of Acid Form of Antibiotic A28695B One hundred mg.of A28695B in the form of the mixed sodium-potassium salt was dissolvedin 25 ml. of dioxane. Twenty ml. of water was added to the resultingsolution and the pH was adjusted to pH 4.0 with hydrochloric acid. Thesolution was concentrated in vacuo in order to remove the dioxane. Theresulting aqueous solution was extracted with an equal volume of ethylacetate and the spent aqueous phase was discarded. The ethyl acetateextract was concentrated to dryness. The dried residue was dissolved ina minimum quantity of warm ethyl ether. The ether solution was held inthe cold in order to allow crystallization of A28695B. The crystals wererecovered by filtration and dried. Yield 87 mg. M.P. 122-124 C.

EXAMPLE 4 Preparation of the sodium salt of A28695A Two hundredmilligrams of A28695A acid, prepared according to the proceduredescribed by Example 2, was dissolved in 10 ml. acetone. To the solutionwas added, with stirring, ml. of water, and the pH of the solution wasadjusted to pH 9.0 with 1N sodium hydroxide. The acetone was slowlyevaporated by placing the solution under a stream of nitrogen. Aprecipitate formed which was recovered and then dissolved in a minimumquantity of diethyl ether. The solution was evaporated to a small volumeand chilled at 5 C. overnight. The resulting crystals were filtered anddried to yield 33 mg. of A28695A sodium salt melting at about 159-160 C.

EXAMPLE 5 Preparation of A28695A ammonium salt Two hundred milligrams ofA28695A acid, prepared according to the procedure in Example 2, wasdissolved in ml. of acetone, and 5 ml. of water was added to thesolution. The pH of the solution was adjusted to pH 9.0

with concentrated ammonium hydroxide. The acetone was slowly evaporatedby placing the solution under a. stream of nitrogen. After the acetonehad been evaporated from the solution, a noncrystalline precipitateformed. The suspension was extracted with an equal volume of diethylether and the resulting ether solution was concentrated to a smallvolume by placing the solution under a stream of nitrogen. Theconcentrated solution was allowed to sit at 5 C. overnight. Acrystalline precipitate formed which was filtered off and dried to yieldmg. of product melting at about 124-125 C.

EXAMPLE 6 Preparation of the sodium salt of A28695B Two hundredmilligrams of the acid form of A28695B, prepared according to theprocedure described in Example 3, was dissolved in 10 ml. of acetone,and 5 ml. of water was added slowly, with stirring, to the resultingsolution. The pH of the solution was adjusted to pH 9.0 with 1N NaOH.The acetone was slowly evaporated by placing the solution under a streamof nitrogen. After the acetone had been evaporated from the solution, acrystalline precipitate began to form. The suspension was allowed tostand at 5 C. overnight in order for the crystallization to be complete.The crystals were filtered and dried to yield 150 mg. of A28695B sodiumsalt melting at about l61162 C.

EXAMPLE 7 Preparation of the ammonium salt of A28695B Two hundredmilligrams of A28695B free acid, prepared according to the proceduredescribed in Example 3, was dissolved in 10 ml. of acetone, and 5 ml. ofwater was added to the resulting solution slowly, with stirring. The pHof the solution was adjusted to pH 9.0 with concentrated ammoniumhydroxide solution. The acetone was evaporated from the solution byplacing the solution under a stream of nitrogen. A crystallineprecipitate began to form after the acetone had evaporated from thesolution. The suspension was allowed to stand at 5 C. to complete thecrystallization. The crystals of the ammonium salt of A28695B wererecovered by filtration and dried to yield 138 mg. of product melting atabout 124- 125 C.

In accordance with the present invention, it has been discovered thatthe A28695 antibiotics, and their physiologically-acceptable salts, thealkali metal, alkaline earth, and basic nitrogen salts, such as ammoniumsalts, are potent anticoccidial agents, which, when administered topoultry and rabbits, arrest the development of coccidia and hence areprophylactic agents in controlling coccidiosis. Suitable alkali metalsalts include the sodium, potassium, and lithium salts, andsodium-potassium mixed salts. Suitable alkaline earth metal saltsinclude the cal cium and barium salts, and the like. These newanticoccidial agents are conveniently fed to poultry or rabbits as afeed component, although the agents may also be suspended in thedrinking water. Thus, novel compositions are provided which compriseA28695 antibiotics intimately admixed with an edible, inert, solidcarrier or diluent to provide a premix for medicated food supplement. Aninert, edible carrier or diluent is one that is nonreactive with respectto the A28695 antibiotics. The carrier or diluent is preferably one thatis or may be an ingredient of animal feed.

The preferred compositions of this invention are feed premixes in whichthe A28695 antibiotics are present in relatively large amounts and whichare suitable for addition to the poultry or rabbit feed either directlyor after an intermediate dilution or blending step. Examples of carriersor diluents suitable for such compositions are animal feed ingredientssuch as distillers dried grains, soybean mill run, alfalfa granules,wheat middlings, corn gluten meal, exfoliated hydrobiotites, corn meal,citrus meal, fermentation residues, ground oyster shells, Attapulgusclay, wheat shorts, molasses solubles, corncob meal, edible vegetablesubstances, toasted dehulled soya flour, soybean feed, soybean mealfeed, antibiotic mycelia, soya grits, kaolin, talc, crushed limestone,and the like. The preferred diluents are soybean products such assoybean mill run and soybean feed, and alfalfa products such as alfalfagranules, as well as corn products such as corn gluten meal and corngrits.

, The compositions can be prepared by intimately dispersing or admixingthe A28695 antibiotics as the pure compounds, a salt thereof, themycelial cake, or the dried broth throughout the solid inert carrier bymethods such as grinding, stirring, milling, or tumbling. In someinstances, the antibiotic may be adsorbed on the carrier by spraying asolution of the antibiotic into a rotating mill containing the carrier.By selecting proper carriers and by altering the ratio of carrier toactive ingredient, compositions of any desired concentration can beprepared.

The feed premixes can be formulated so that the total active ingredientis present within the range of percent to 80 percent by weight and thediluent or carrier present is correspondingly within the range of 95percent to percent by weight of the premix. The preferred ratio is about8 percent by weight of antibiotic A28695A to about 92 percent by weightof the diluent. The preferred ratio for antibiotic A28695B is aboutpercent of antibiotic to about 70 percent by weight of the diluent. Thepremixes may be further diluted with an animal feed supplement or may beadded directly to an animal feedstulf in order to provide a suitablemedicated feedstutf which can be eaten directly by the poultry orrabbits.

The feed supplement may be further diluted with mate rials such as cornmeal, corn grits, corn gluten meal, or

soybean meal before being incorporated in the animal feed. This dilutionserves to facilitate uniform distribution of the anticoccidial agents inthe finished feed. The finished feed is one that contains a source offat, protein, carbohydrate, minerals, vitamins and other nutritionalfactors.

In the treatment of coccidiosis infections, relatively low levels of theA28695 antibiotics in poultry or rabbit feed are sufficient to affordthe poultry or rabbits good protection against coccidiosis. One of theantibiotics, antibiotic A28695A or A28695B, or the mixture ofantibiotics from which they can be separated, identified as A28695,isadministered to chickens in an amount equal to about 0.00125 percentto about 0.03 percent by weight of the daily feed intake. The preferredrange for the mixture, antibiotic A28695, is from about 0.0025 percentto about 0.02 percent by weight of the daily feed intake. The preferredrange for antibiotic A28695A is from about 0.0025 to about 0.01 percent,with the optimum results being obtained when from about 0.0025 to about0.005 percent (22.7-45.4 g./ton) is incorporated into the poultry feed.The preferred range for antibiotic A28695B is from about 0.015 to about0.03 percent by weight of the antibiotic incorporated into the poultryfeed, with 0.02 percent (181.6 g./ton) of antibiotic A28695B being theoptimum level for that antibiotic. Antibiotic A28695A, when administeredin an amount equal to about 0.00125 percent to about 0.005 percent byweight of the rabbit feed, is efiicacious as an anticoccidial agent inrabbits. About twice as much antibiotic A28695B is necessary in rabbitsto accomplish the same result as with A28695A.

The most advantageous dosage level will, of course, vary somewhat withparticular circumstances such as the type and severity of the coccidialinfection to be treated, the daily feed intake of the birds or rabbits,and the like.

Prior to the administration of the medicated feed to 14 the poultry orrabbits, the premix is uniformly dispersed in the animal feed bysuitable mixing or blending procedures.

In the above discussion of the invention, emphasis has been placed onsolid compositions wherein the active ingredient is mixed with an ediblecarrier in a premix, or in the final poultry or rabbit feedstuif.

An alternative method of treatment is to administer a suspensioncontaining a theraputically-elfective amount of the mixture of A28695antibiotics, or antibiotic A28695A or A28695B, respectively, or a saltthereof, in the drinking water of the poultry. The quantity of theanticoccidial agent which can be administered in this fashion is, ofcourse, limited by the solubility of the antibiotics in water or by thequantity that can be suspended in the water without undue settling.Emulsifiers or surfactants can be employed in order to increase theamount of antibiotic which may be suspended in solution.

In treating poultry according to the method of this invention, theantibiotic can be administered starting with very young chicks. Thus,l-day-old broiler cockerels are started on the medicated feed containingthe A28695 anti biotic. This procedure applies for broilers, roasters,fryers, and for replacement stock for layer or broiler breeder flocks.Broiler birds are maintained on the medicated feed of this inventionthroughout their life. Replacement stock for layer flocks are maintainedon this medicated feed for about 14 to 22 Weeks.

Generally speaking, the antibiotic, or one of its salts, is added to aconventional basal ration which can comprise the following ingredients:meat and bone scrap; fishmeal; Vitamin B poultry by-product meal;dehulled soybean oil meal; dehydrated alfalfa meal; corn gluten meal;pulverized oats; ground barley; cornmeal; wheat middlings; dried grainand whey fermentation solubles; methionine hydroxy analogue calcium;riboflavin; calcium pantothenate; choline chloride; niacin; animal fat;menadione sodium bisulfite; Vitamin E supplement; butylated hydroxytoluene; Vitamin A palmitate; D-activated animal sterol; calciumcarbonate; defluorinated phosphate; salt; calcium iodate; manganeseoxide; zinc oxide; cobalt hydroxide; and cobalt carbonate.

The efficacy of the A28695 antibiotics as anticoccidial agents in bothchickens and rabbits has been demonstrated in a series of experiments.In general, the experiments are carried out as described hereinafter.

For the studies in chickens, groups of about five 8-dayold chicks arefed a standard diet containing therein a uniformly dispersed A28695antibiotic. After being on this ration for about 48 hours, each bird isinoculated with sporulated oocysts of the particular species of Eimeriabeing tested.

Other groups of five, 8-day-old chickens are fed a standard diet whichdoes not contain an A28695 antibiotic. Some of these latter groups arealso inoculated with Eimeria after 48 hours and they serve as infectedcontrols. Others of these latter groups are not inoculated with Eimeriaand they serve as normal controls.

The results of the treatment are evaluated 7 days after the inoculation.The birds are weighed, sacrificed, and examined for evidence ofcoccidial lesions. The percent reduction in lesion score is calculatedby subtracting the average lesion score of the treated group from theaverage lesion score of the infected control group, dividing thisdifference by the average lesion score of the infected control group,and multiplying the quotient by 100.

The percent weight gain is calculated using the weight gain of normalcontrols as percent.

b iIhe experiments and the results are set forth hereine ow.

EXAMPLE 1 The efiicacy of the A28695 antibiotics A and B in controllingcoccidiosis in chickens was determined by adding each antibiotic to thefeed of chickens infected with a combined infection of Eimeria tenella,Eimeria ne'catrix, Eimeria maxima and Eimeria acervulina.

The results are set forth in the following Table I.

TABLE I Activity of A28605 antibiotics against eoccidiosis in chickensPercent.

Level in Reduction in feed, lesion scores" percent by Mor- Weight TestGroup weight tality gain" Intestinal Cecal A28695B 0. 02 0 90 100 96Infected controls 36 O 0 Normal controls 0 100 Normal controls taken as100%. Compared with infected controls.

EXAMPLE 2.

A test of the efiicacy of the A28695 antibiotics A and B against Eimeriatenella in chickens was carried out and the results are shown in thefollowing Table II.

The efficacy of antibiotic A28695A in controlling coccidiosis inchickens infected with Eimeria necatrix was determined by adding theantibiotic to the feed of chickens.

The chickens, 8-day-old broiler cockcrels, were divided into groups offive birds per cage, four cages per treatment. The birds were fed astandard diet containing antibiotic A28695A uniformly dispersed therein.Forty-eight hours after the medication was commenced, each of the birdswas inoculated with sporulated oocysts of E. necatrix.

Other groups of five, 8-day-old broiler cockerels were fed the standarddiet which did not contain antibiotic A28695A. Some of these groups werealso inoculated with E. necatrix after 48 hours and served as infectedcontrols. Those not infected served as normal controls. The test wasterminated 7 days after the inoculation. The birds were weighed,sacrificed and examined for evidence of coceidial lesions. The resultswere calculated as described, supra, and are shown in Table III, whichfollows.

Normal controls taken as 100%. "Compared with infected controls.

1 6 EXAMPLE 4 The efficacy of antibiotic A28695A in controllingcoccidiosis in chickens infected with Eimeria brunetti was determined byadding the antibiotic to the feed of the chickens.

The chickens, S-day-old broiler cockerels, were divided into groups offive birds per cage, four cages per treatment. The birds were fed astandard diet containing antibiotic A28695A uniformly dispersed therein.Forty-eight hours after the medication was commenced, each of the birdswas inoculated with sporulated oocysts of E. brunetti.

Other groups of five 8-day-old broiler cockerels were fed the standarddiet which did not contain antibiotic A28695A. Some of these groups werealso inoculated with E. brunetti after 48 hours and served as infectedcontrols. Those not infected served as normal controls. The test wasterminated 7 days after inoculation. The birds were weighed, sacrificed,and examined for evidence of coccidial lesions. The results are setforth in Table IV, which follows.

TABLE IV Percent Reduction Weight lesion Treatment Diet Mortality gain*score E. brunctti Infected controls 0 Normal control. 0

*Normal controls taken as Compared with infected controls. "Less than30%.

EXAMPLE 5 The efficacy of antibiotic A28695A in controlling coccidiosisin chickens infected with Eimeria mivati was determined in the samemanner, with the same age and type of chickens as described in Example4. The dosage levels of antibiotic A28695A were the same. The testresults are set forth in Table V, which follows.

Normal controls taken as 100%. "Compared with infected controls. *Lessthan 30%.

EXAMPLE 6 Following the same procedure as set forth in the precedingexamples, 8-day-old broiler cockerels were used to test the efficacy ofantibiotic A28695A in controlling coccidiosis in chidkens infected with*Eimeria acervulina. The results are set forth in Table VI, whichfollows.

*Normal controls taken as 100%. "Compared with infected controls.

EXAMPLE 7 Following the same procedure set forth in the precedingexamples, S-day-old broiler cockerels were used to determine theefiicacy of antibiotic A28695A in controlling coccidiosis in chickensinfected with Eimeria maxima.

The results are set forth in Table VII, which follows.

TABLE VII Percent Reduction Weight lesion Treatment Diet Mortality gain*score E. mazima Infected controls 0 Normal controls. 0

"Normal controls taken as 100%. "Compared with infected controls.

EXAMPLE 8 The efiicacy of the mixture of antibiotics A28695, as isolatedfrom the fermentation, in controlling coccidiosis in chickens infectedwith 'Eimeria tenella was determined by adding the antibiotic to thefeed of chickens. The test material is a mixture and is so identified inthe table to follow.

The chickens, S-day-old broiler cockerels, were divided into groups offive birds per cage, two cages per treatment. The birds were fed astandard diet containing antibiotic A2869 uniformly dispersed therein.Forty-eight hours after the medication was commenced, each of the birdswas inoculated with sporulated oocysts of E. tenella.

Other groups of five, 8-day-old broiler cockerels were fed the standarddiet which did not contain antibiotic A28695. Some of these groups werealso inoculated with E. tenella after 48 hours and served as infectedcontrols. Those not infected served as normal controls. The test wasterminated seven days after the inoculation period. The birds wereweighed, sacrificed and examined for evidence of coccidial lesions. Theresults were calculated as previously described and are shown in TableVIII which follows.

Normal controls taken as 100%. Compared with infected controls. "*Lessthan 30%.

'18 EXAMPLE 9 The efiicacy of antibiotics A28695 (the mixture),antibiotic A28695A, and antibiotic A28695B, in controlling coccidiosisin chickens infected with Eimeriai tenella, was determined by adding therespective antibiotics to the feed of chickens. V

The chickens, 8-day-old broiler cockerels, were divided into groups offive birds per cage, three cages per treat ment. The birds were fed astandard diet containing antibiotic A28695, antibiotic A28695A, orantibiotic A28695B uniformly dispersed in the diet. Forty-eight hoursafter the medication was commenced, each' of the birds was inoculatedwith sporulated oocysts of E. tenella 'Other groups of five, 8-day-oldbroiler cockerels were fed the standard diet which did not contain anyof the above-named antibiotics. Some of these groups were alsoinoculated with E. tenella after 48 hours and served as infectedcontrols. Those not infected served as normal controls. The test wasterminated seven days after the inoculation, The birds were weighed,sacrificed and examined for evidence of coccidial lesions. The resultswere calculated as described previously and are shown in Table IX whichfollows.

Normal controls taken as Compared with infected controls. '"Less than30%.

EXAMPLE 10 The efiicacy of antibiotic A28695A in controlling coccidiosisin rabbits infected with Eimeriai stiedae, which affects the liver, andin controlling naturally-occurring intestinal coccidia, was determinedby adding the antibiotic to the feed of the rabbits.

The medicated feed for administration to the rabbits was prepared byfirst preparing a premix. The most concentrated premix contained 1.136g. of antibiotic A28695A per lb. The antibiotic was blended withsolventextracted soybean meal by addition of small increments of theantibiotic to the carrier in a large mortar. The antibiotic and thesoybean meal were finely ground, blended,

and passed through a number 12 screen to produce the.

final premix. It was utilized to produce a feed containing 0.005 percentantibiotic A28695A.

The second premix contained 0.578 g. of antibiotic A28695A per lb. ofpremix. It was prepared by blending that weight of A28695A withsolvent-extracted soybean meal by addition of small increments of thecompound to the carrier in a large mortar. The mixture was finely groundand blended and passed through a number 12 screen for the final product.This premix was utilized to prepare feed containing 0.0025 percentantibiotic A28695A.

In a similar manner, 0.284 g. of antibiotic A28695A was blended withsolvent-extracted soybean meal as described above. This premix wasutilized to prepare feed containing 0.00125 percent antibiotic A28695A.

These premixes were then individually mixed with Rockland rabbit ration(a standard, commercially available rabbi t ration), .and the medicatedmash thus produced was formed into pellets for feeding to the rabbits.

Susceptible young rabbits, four to eight weeks, old, were divided intogroups of six rabbits (3 rabbits/cage) per treatment. The rabbits werefed a standard diet containing antibiotic A28695A uniformly dispersed inthe diet. Four days after the medication wascommenced, each of therabbits was inoculated orally with sporulated oocysts of E. stiedae.

Another group of six of the susceptible rabbits was fed the standarddiet which did not contain any of the antibiotic A28695A. This group wasalso inoculated with E. stiedae four days after the medication wascommenced, and this group served as the infected control. Other groupsof six rabbits each were fed a standard diet containing no antibioticA28695A, and these rabbits were not inoculated withE. stiedae. Thesegroups served as the uninfected (as to E. stiedae) controls.

The test was terminated 26 days after the inoculation. These rabbitswere weighed, sacrificed, and examined for evidence of coccidiallesions.

' During the test, the feces of six rabbits per test group (2 cages of 3rabbits each) were collected on the day of inoculation and pooled. Thefeces were then collected twice weekly for 14 days and then daily for14. days. The feces were examined for the presence of the oocysts ofintestinal coccidia as well as of E. Stiedae. The intestinal species ofcoccidia identified by examination of the feces were E. perforans, E.magna, and E. Neoleporis.

At necropsy, the weight of the liver of each rabbit was determined, andthe liver weights were calculated as a percentage of total average bodyweight. Severe infections with E. stz'ea'ae cause enlargement of thelivers. The normal liver is 23% of the total body weight.

The rabbits on the highest medication level showed a marked decline inweight which was probably due to failure to eat the medicated pellets.All the rabbits in the control group were severely infected withintestinal coccidia at the start of the test, the infection beingnatural, not artificial.

Table X, which follows, lists the results of the test. Column 1 'liststhe percent by weightof antibiotic inthe feed; columns 2 through 5, theaverage gross weights of the rabbits on the designated test days; column6, the average gross weights of the rabbits at necropsy; column 7, theaverage liver weight; and column 8, the relation of liver weight tototal body weight, expressed as percent.

Day inoculated with E. Stiedae.

"Day of necropsy.

Table XI, which follows, sets forth the number of oocysts of intestinalcoccidia and of E. slicdae found in the feces from the six rabbits pertest group on examination on specified days beginning on the day ofinoculation and continuing through the day of termination of the test.

The results show anti-biotic A2869SA to be elfective against both theliver form as well as intestinal forms of coccidia.

It should be noted that the infection by the intestinal coccidia wasnaturally-occurring. It is cyclical and this explains the decrease anddisappearance of oocysts of the intestinal coccidia in the feces andthen the reappearance of those oocysts at a later date in the feces ofthe animals which received none of the test compound in their feed. Italso explains the variation in the intestinal coccidia oocysts found inthe feces of the groups of control animals. These control animals werenaturally infected with intestinal coccidia. They were not inoculatedwith oocysts of Eimeria stieaae, but became infected through proximityof cages.

TABLE XI 00cyst passage per test group Percent antibiotic IntestinalEimeria A28695A v coccidia stz'cdae Test day N o. 4*

Test day No. 22

0.005 0.0026 0.00125. 0 ControL 600 1, 200 D0; 200 68, 200

4 Test day No. 23

137, 800 Control. 8, 200 Do 36, 000

Test day No. 24

Test day No. 25

1, 240, 000 Control 11,2

TABLE XL-Continued Oocyst passage per test group Percent antibioticIntestinal Eimeria A28695A coccidia stiedae Test day No. 26

1, 000, 000 Control 11, 800 D 800 37, 800

Test day No. 27

1, 500, 000 Control 2, 600 4,

Test day No. 28

Test day No. 29

Day of inoculation.

It will be apparent to those skilled in the art that the anticoccidialagents of this invention may be used either alone or in combination withone or more other anticoccidial agents. That is, poultry can be treatedwith compositions having antibiotic A28695, A28695A, or A28695B, or oneof the salts as the sole anticoccidial agent, or treatment can beinitiated wherein one of the antibiotics or one of the salts and one ormore other anticoccidial agents are administered concurrently. For suchpurposes, antibiotic A28695, A28695A, or A28695A can be admixed with oneor more other anticoccidial agents including, but not limited to, thefollowing:

3,5-dinitro-0-toluamide;

3,5-dichloro-2,5-dimethyl-4-pyridinol;

1, (4-amino-2-n-propyl-5-pyrimidinylmethyl-Z-picolinium chloridehydrochloride;

ethyl 4-hydroxy-6,7-diisobutoxy-3-quinolinecarboxylate;

7-benzyloxy-6-n-butyl-3-methoxycarbonylquinol-4-one;

ethyl 4-hydroxy-6-n-decyloxy-7-ethonoxyquinoline- S-carboxylate;

2,4-diamino-5- (3 ,4-dimethoxybenzyl) -pyrimidine;2-chloro-4-nitrobenzamide;

sulfaquinoxaline; other sulfa compounds;4,4-dinitrocarbanilide-2-hydroxy-4,6-dimethylpyrimidine complex;3,3'-dinitrodiphenyldisulfide; arsanilic acid;

3-amino-4-hydroxyphenylarsonic acid; S-nitrofurfural semicarbazone, andthe 6,7-dialkoxy-4-hydroxyquinoxaline-3-carboxy1ic acid esters.

In using combined therapy, therapeutically-effective amounts ofantibiotic A28695, A28695A, or A28695B, and another anticoccidial agentare incorporated into the compositions discussed herein above. It isobvious to those skilled in the art that combined therapy may benecessary when various species of the protazoan parasite of the genusEimeria are involved.

We claim:

1. A method of treating and preventing coccidiosis in poultry andrabbits which comprises administering orally to poultry and rabbits ananticoccidially-elfective amount of antibiotic selected from the groupconsisting of A28695, A28695A, A28695B, and the sodium salt, potassiumsalt, sodium-potassium mixed salt, or ammonium salt thereof.

2. The method of Claim 1 wherein antibiotic A28695 is administeredorally to poultry at the rate of from about 0.00125 percent to about0.03 percent by weight of the daily feed intake.

3. The method of Claim 1 wherein antibiotic A28695 is administeredorally to poultry at the rate of from about 0.0025 percent to about 0.02percent by weight of the daily feed intake.

4. The method of Claim 1 wherein antibiotic A28695A is administeredorally to poultry at the rate of from about 0.0025 to about 0.01 percentby weight of the daily feed intake.

5. The method of Claim 1 wherein antibiotic A28695B is administeredorally to poultry at the rate of from about 0.015 to about 0.03 percentby weight of the daily feed intake.

6. The method of Claim 1 wherein antibiotic A28695A is administeredorally to rabbits at the rate of from about 0.00125 percent to about0.005 percent by weight of the daily feed intake.

References Cited Miller, The Pfizer Handbook of Microbial Metabolites,McGraw-Hill Book Co., Inc. New York, N.Y., 1961, pages 573 and 575.

JEROME D. GOLDBERG, Primary Examiner US. Cl. X.R. 424122

1. A METHOD OF TREATING AND PREVENTING COCOCIDIOSIS IN POULTRY ANDRABBITS WHICH COMPRISES ADMINISTERING ORALLY TO POULTRY AND RABBITS ANANTICOCCIDALLY-EFFECTIVE AMOUNT OF ANTIBIOTIC SELECTED FROM THE GROUPCONSISTING OF A28695, A28695A, A28695B, AND THE SODIUM SALT, POTASSIUMSALT, SODIUM-POTASSIUM MIXED SALT, OR AMMONIUM SALT THEREOF.